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. 2020 Apr 23;58(5):e02088-19.
doi: 10.1128/JCM.02088-19. Print 2020 Apr 23.

Differentiation of Community-Associated and Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates and Identification of spa Types by Use of PCR and High-Resolution Melt Curve Analysis

Seyed A Ghorashi  1   2 , Jane Heller  3   2 , Quincy Zhang  3   2 , Shafi Sahibzada  3   2   4
Affiliations

Differentiation of Community-Associated and Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates and Identification of spa Types by Use of PCR and High-Resolution Melt Curve Analysis

Seyed A Ghorashi et al. J Clin Microbiol. .

Abstract

Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. The capability of PCR followed by high-resolution melt (HRM) curve analysis for the detection of community-associated and livestock-associated MRSA strains and the identification of staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans, and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A nonsubjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings, and result data were reanalyzed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies.

Keywords: MRSA; PCR; high-resolution melt curve analysis.

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Figures

FIG 1
FIG 1
Conventional and normalized melt curve analysis of MRSA isolates. (a) Conventional and (b) normalized melt curve analysis of PCR amplicons from CA-MRSA (ST93) and LA-MRSA (ST398) isolates. All LA-MRSA isolates produced a single peak (mean 82.3°C), while CA-MRSA isolates produced one peak at higher temperature (mean 83.8°C) and a shoulder peak at lower temperature. The P071 (ST30) isolate was distinct from the ST93 and ST398 isolates.
FIG 2
FIG 2
Differentiation of MRSA and MSSA isolates using conventional and normalized melt curve analysis. (a) Conventional and (b) normalized HRM curve analysis of PCR amplicons for CA-MRSA (ST93) (red), LA-MRSA (ST398) (blue), and MSSA (green) isolates.
FIG 3
FIG 3
Comparison of the distribution of GCPs from CA-MRSA (ST93) and LA-MRSA (ST398) isolates by individual value plot when H029 and P030, respectively, were used as reference genotypes.
FIG 4
FIG 4
Comparison of the distribution of GCPs from MRSA and MSSA isolates by individual value plot when the MSSA isolate (20-L) was used as the reference genotype.
FIG 5
FIG 5
Phylogenetic relationship of spa types and MRSA/MSSA isolates based on the nucleotide sequence of the spa gene.
See this image and copyright information in PMC

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